The purpose of this research is to elucidate the mechanism by which maleylacetone cis-trans isomerase together with glutathione (GSH) catalyzes cis-trans isomerization of maleylacetone and maleylacetoacetate. Mechanisms for the enzymatic reaction are proposed and experimental tests are suggested. The most important conclusions from recent experiments are: 1) GSH's role in the enzymatic reaction is to provide nucleophilic catalysis which leads to a GS-dienediol, coenzyme-substrate, intermediate. 2) There is a catalytically important enzymic sulfhydryl group as shown by N-ethylmaleimide inhibition and its binding to a cysteine residue. A mechanism to account for these observations suggests that maleylacetone and enzyme react to form a thiohemiketal and then GSH carries out an SN2' reaction to form the GS-dienediol intermediate which is capable of facile internal rotation followed by reketonization with GSH expulsion. The thiohemiketal mechanism will be tested in three ways: a) a bifunctional inhibitor, GS (CH2)n-N-maleimide (n equals 1-4) will be used to determine the distance between the enzymic sulfhydryl and the sulfhydryl of the coenzyme. This distance will be compared with that expected for the thiohemiketal mechanism. b) 1H-nmr cross saturation experiments will be used to determine the presence of an enzyme-substrate thiohemiketal complex. c) A substrate analogue proposed as a suicide inhibitor will be synthesized and tested. An alternative mechanism leading to the same GS-dienediol intermediate is suggested to take place by enzymic protonation of the substrate's carbonyl oxygen in advance of or in concert with GSH-nucleophilic attack. A substrate analogue is proposed to trap the proton added by the enzyme and provides examination of this mechanism. Other analogues are proposed for synthesis and testing to define the conformation of the substrate when it is associated with enzyme.